AU2007273886A1 - Bisnaphthalimidopropyl derivative compounds with anti-parasite and anti-cancer activity - Google Patents

Description

WO 2008/007262 PCT/IB2007/052311 1 Description BISNAPHTHALIMIDOPROPYL DERIVATIVE COMPOUNDS WITH ANTI-PARASITE AND ANTI-CANCER ACTIVITY Technical domain of the invention [1] The new bisnaphthalimidopropil derivatives, preparation process thereof, pharma ceutical composition comprising them and their use in cancer and parasitic diseases, namely leishmaniasis, trypanosomiasis and malaria, with pharmaceutical industry ap plication. [2] The present invention referred to a preparation process of the new bisnaphthalim idopropil derivative compounds and pharmaceutical compositions comprising them. These compounds have a role in the growth inhibition of the parasite protozoa Leishmania infantum and cytotoxic properties on cancer cells. Previous State of art [3] Naphthalimido derivatives exhibit considerable potential as cytotoxic agents for cancer chemotherapy (Brana et al., 2001). We previously reported the synthesis and biological activities of a novel series of bisnaphthalimidopropyl polyamines compounds (Kong et al., 2000). Subsequent work revealed the presence of the bisnaphthalimidopropyl functionality to be essential for optimum biological activity since the presence of an oxygen atom in the alpha-position of the naphthailimido ring tends to reduce activity (Pavlov et al., 200 1). [4] Majority research traditionally focused on the modification of the naphthalimido rings to enhance anticancer activities through increased DNA binding and cleavage. For example, acenaphthalimide was introduced into the naphthalimide chromophore to increase the solubility of the bisnaphthalimide compounds (Patten et al., 1992). Furan heterocycles were added to the naphthalimide chromophore and those compounds exhibited strong DNA binding properties with toxicity to CEM leukaemia cells to be in the nanomolar concentration (Brana et al., 1995). Pyrazine heterocycles have also recently been fused to naphthalimides and those pyrazino-naphthalimides exhibited in vitro toxicity with IC 5 o values ranging from 0.002 to 7.8 pM after 72 hour treatment m cancer HT 29, HeLa, and PC 3 cells (Bailly et al., 2003). [5] However, in our laboratory we have developed bisnaphthalimidopropyl fragments linked to natural polyamines such as putrescine, spermidine and spermine. The spermidine and spermine derivatives exhibited enhanced aqueous solubility while maintaining good biological activity (Carrasco et al., 2003 and Kong et al., 2003). In MCF 7 breast cancer cells, compounds were observed within the cell nuclei after 6 and 12 hour drug exposure, with transport being potentially energy dependent (Dance et al. , 2005). Within MCF7 cells, the bisnaphthalimidopropyl compounds inflicted sig nificant quantitative DNA damage. We also found for the first time that bisnaph thalimido propyl derivatives exert significant anti-proliferative effects on the life cycle WO 2008/007262 PCT/IB2007/052311 2 of Leishmania infantum, the causative agent of visceral leismaniasis and these drugs also induced the death of promastigotes by apoptosis (Tavares et aL, 2005). [6] In the leishmaniasis the first line chemotherapy is restricted to the use of pentavalent antimony derivatives (Murry et al., 2001). As a consequence of the long course of the therapy, the adverse reactions appear and the resistances induce the search of new drug more efficient. The efficacy of the treatment be also comprised in immunodeficiency situation, namely in the co infection Leismania/HIV. [7] The natural polyamine, putresceine, spermidine, and spermine are present in most of the eukaryotic cells and have an important role on proliferation and cellular differ entiation (Muller et al., 2001). In the case of trypanosomatids the polyamines have an additional role in the endogenous oxi-redox equilibrium by the trypanothione compound [N1, N8-bis (glutathione) spermidine]. These molecules and enzymatic reactions have been considered as a good drug targets (Fairlamb and Cerami, 1992; Barrete et al., 1999). Interfere with the regulatory function of the polyamines have been a strategy to search for new compounds with anti-cancer and anti-parasitic activity. Polyamine synthesis inhibitors as a DFMO (alpha-difluormetylornitine) showed to be active against different parasite stages of Plasmodium sp. This parasite has a bifunctional enzyme with ornithine and S-adenosylmethionine decarboxilase activity, that became attractive as a drug target since it is absent in the host cells (M0ler et al, 2001). [8] Interfere with regulatory function of polyamines became a strategy to search the efficacy compounds with anti-cancer and anti-parasitic activity. [9] In this invention we report the synthesis of analogues bisnaphthalimidopropyl di and tri-amines: BNIPDapen, BNIPDhex, BNIPDahep, BNIPDaocet, BNIPDanon, BNIPDadec, BNIPDadod, BNIPDpta and BNIPDeta based on our leading compounds BNIPPut (I) and BNIPSpd (XI) (spermidine derivative) with modi fication of the central chain. The modification consists of different alkyl length of the central chain with 2 or 3 nitrogen atoms, thus modulating the number of positive charges in the molecules. We also discuss the in vitro cytotoxic properties of these newly synthesised compounds in colon cancer cells (CaCo-2) and parasites ( Leishmania infantum, promastigotes). The modifications include the power, spe cificity, oral biodistribution, penetration into target tissue and action duration, concerning the new compounds from the general formulation A. [10] WO 2008/007262 PCT/IB2007/052311 3 0 /0N H H 0 Point of div ersity 0 BtHIPDiamuinoalkyl compounrds 0 ?H/ BNIPDabutN BNiP~apen H 2HBr H 0 - N ' N r~~~ N ~~' BNIPDaperk ' H 21-1r H 0 0 1 0 BNIPOahex H 2HBr 0 N ~-~ ~~' ~N~~h - IV BNIP Dabep H ) B NIP Da Oct N 2HBr H B NIP Danton Ni BNIP Ldec OV1 H )Pad BNIP~adod _ ~ ~ NrN-x N~VI ~ { H 2HBr 0) WO 2008/007262 PCT/IB2007/052311 4 BNIPTriamninoalkyl compounds 0 0 0 ox / \ N N N N Ix BNIPDpta H H H ~ 0 H N IPD eta r 0 H 3HBr H ' x1 HH wherein: the alkyl central chain has modifications in the length and in the introduction of nitrogen atoms. [11] The compounds showed in formulation A are: 1. Bisnaphthalimidopropylputrescine - BNIPPut (I) 2. Bisnaphthalimidopropyldiaminopentane - BNIPDapen (II) 3. Bisnaphthalimidopropyldiaminohexane - BNIPDahex (III) 4. Bisnaphthalimidopropyldiaminoheptane - BNIPDahep (IV) 5. Bisnaphthalimidopropyldiaminooctane - BNIPDaoct (V) 6. Bisnaphthalimidopropyldiaminononane - BNIPDanon (VI) 7. Bisnaphthalimidopropyldiaminodecane - BNIPDadec (VII) 8. Bisnaphthalimidopropyldiaminododecane-BNIPDadod (VIII) 9. Bisnaphthalinidopropyldipropiltriamine - BNIPDpta (IX) 10. Bisnaphthalimidopropyldietiltriamine-BNIPDeta (X) 11. Bisnaphthalimidopropylespermidine - BNIPSpd (XI) [12] Another aspect of the invention concern the preparation process, pharmaceutical formulation made in combination of one compound of formula II, III, IV, VI, VII, VIII, IX, X with a vehicle or safety pharmaceutical excipient. Description of the invention [13] The synthesis of the protected compounds in this invention was based on methods previously described by our group (Kong et al., 2003). The bisnaphthalimido compounds with linker chain containing 2 nitrogens were previously synthesised by WO 2008/007262 PCT/IB2007/052311 5 simply reacting the corresponding alkyltetraamine with 1,8-naphthalic anhydride (Brana et al., 1995). In order to introduce more heteroatoms in the linker chain, N alkylation reaction was chosen according to a Kong modified method (Kong et al., 1998). The common intermediate for the synthesis of different compounds was tolu enesulfonyloxypropylnaphthalimide. This was prepared by first reacting 1,8-naphthalic anhydride with aminopropanol to give N-(3-hydroxypropyl)naphthalimide which upon reaction with tosyl chloride gave toluenesulfonyloxypropylnaphthalimide, with 60% yield. To obtain the bisnaphthimide the polyamine used depends on the compound to synthesise and were first protected with 2, 4, 5- trimethylsulphonyl chloride (Mts-Cl) in pyridine followed by their N-alkylation with toluenesulfonyloxypropylnaph thalimide produced the bisnaphthalimide derivative protected. The deprotection was obtained with hydrobromic acid/glacial acetic acid in dichloromethane to give the re spective derivative as their hydrobromide salts. [14] CaCo-2 cells (ECACC, 86010202) were obtained from the European Collection of Cell Cultures. All reagents were purchased from Aldrich, Fluka and Lancaster and were used without purification. TLC was performed on Kieselgel plates (Merck) 60 F 254 in chloroform: methanol (97:3 or 99:1). Column Chromatography was done with silica gel 60, 230-400 meshes using chloroform and methanol as eluent. FAB-mass spectra were obtained on a VG Analytical AutoSpec (25Kv) spectrometer; EC/CI spectra were performed on a Micromass Quatro II (low resolution) or a VG Analytical ZAB-E instrument (accurate mass). 1H and 13 C NMR spectra were recorded on a JEOL JNM-EX90 FT NMR spectrometer. [15] BNIPSpd and BNIPPut were synthesised according to our methods previously reported (Kong et al, 2003 and Tavares et al., 2005). [16] CvtotoxicitV studies [17] Cytotoxity was evaluated for CaCo-2 colon carcinoma using the MTT assay with protocols appropriate for the individual test system.

11

,

13 CaCo-2 cells were maintained in Earle's Minimum Essential Medium (Sigma), supplemented with 10% fetal calf serum (Biosera), 2mM L-glutamine (Sigma), 1% non-essential amino acids (Sigma), 100 IU mL- 1 penicillin and 100 m g mL- 1 streptomycin (Sigma). Exponentially growing cells were plated at 2 x 104 cells cm 2 into 96-well plates and incubated for 24 h before the addition of drugs. Stock solutions of compounds were initially dissolved in 20% DMSO and further diluted with fresh complete medium. [18] After 24 and 48 h of incubation at 37 'C, the medium was removed and 200 pl of MTT reagent (1 mg/mL) in serum free medium was added to each well. The plates were incubated at 37 'C for 4 h. At the end of the incubation period, the medium was removed and pure DMSO (200 pl) was added to each well. The metabolized MTT product dissolved in DMSO was quantified by reading the absorbance at 560 nim on a micro plate reader (Dynex Technologies, USA). IC 50 values are defined, as the drug concentrations required to reduce the absorbance by 50% of the control values. The IC WO 2008/007262 PCT/IB2007/052311 6 5o values were calculated from the equation of the logarithmic line determined by fitting the best line (Microsoft Excel) to the curve formed from the data. The IC 50 value was obtained from the equation for y=50 (50% value). [19] Leishmania infantum (clone MHOM/MA67ITMA-P263) promastigotes transfected with reporter gene that encode to the luciferase enzyme (Roy et al., 2000) were grown at 27 'C in RPI medium (Gibco) supplemented with 10% of heat inactivated fetal bovine serum (FBS- Gibco), 2mM L-glutamine (Gibco), 20mM Hepes (Gibco), 1OOU/ml penicillin (Gibco) and 100 p/ml streptomycin (Gibco). The parasites (106/ml) in the logarithmic phase (2 days of culture) were incubated with a serial range of con centrations of each drug for 3 days at 27 'C and the growth of parasites was de termined by using the luciferase activity using luciferin as subtract. [20] The axenic amastigote of Leishmania infantum (clone MHOM/MA671TMA-P263) transfected with reporter gene that encode to the luciferase enzyme (Roy et al., 2000) were grown at 37'C with 5% CO 2 in a cell-free medium called MAA( medium for ax enically grown amastigote). The medium MAA/20, consisted of modified medium 199 (Hanks' balanced salts) supplemented with 0.5% soya trypto-casein, 15mM D-glucose, 5mM L-glutamine, 4mM NaHCO 3 , 0.023mM bovine hemin, 25mM HEPES final pH, 6.5 and 20% inactivated fetal calf serum. The parasites were incubated with different concentrations during 3 days at 37 0 C with 5% CO 2 . The growth of the parasites was done by measuring the luciferase activity using luciferin as a substract. The intra cellular amastigotes of L. infantum were cultured in a macrophage differentiated human leukemia monocyte cell line (THP- 1 cells). The THP-1 cells were differentiated during 2 days with 20ng/ml of PMA in RPMI- 1640 medium supplemented with 10% FCS, 2mM glutamine, 100 IU of penicillin/ml and 100 m g/ml of streptomycin. The non differentiated cells were washed with prewarmed medium and the adherent cells sinfected with luciferase-expressing axenic amastigotes at a parasite/macrophage ratio of 3:1 for 4 h at 37 'C with 5% CO 2 . Noninternalized parasites were removed and serial dilutions of each drug were made in the RPMI medium supplemented with 10%FCS. After 3 days of drug exposure, wells containing adherent differentiated THP-1 cells were washed and luciferase activity was determined. Results [21] Chemistrv [22] The synthetic strategy adopted to synthesise bisnaphthalimidopropyl derivatives BNIPDapen, BNIPDhex, BNIPDahep, BNIPDaoct, BNIPDanon, BNIPDadec, BNIPDadod, BNIPDpta, BNIPDeta based on methods previously developed in our laboratory (Kong et al, 2000). Protection and activation of all the di- and tri- amines were carried with mesitylene chloride in pyridine at room temperature to give compounds 1-5 in high yield. N-alkylation of the latter compounds with 0 tosylpropylnaphthalimide 6 with Ceasium carbonate in anhydrous DMF, afforded the fully protected Bisnaphthalimidopropyl derivatives which upon deprotection with hy- WO 2008/007262 PCT/IB2007/052311 7 drobromic acid/glacial acetic acid in CH 2 C1 2 gave BNIPDapen, BNIPDhex, BNIPDahep, BNIPDaoct, BNIPDanon, BNIPDadec, BNIPDadod, BNIPDpta, BNIPDeta in yield varying from 50-70%. [23] Scheme 1. Synthetic strategy for the synthesis of Bisnaphthalimidopropylal kylamines derivatives.

H

2 NX'- (CH 2 )r NH2 Alkyldiamines in pyridine at RT for 2 hours Mts x (C 2) Ms 0H H n=3,4,6,7,0,10 0 1 i1. DMF, Cs 2 003for 12 hr at 404C Mts ~ 2o CH 2

C

2 , 20% HBrIgalcial CH 00 OH overnight at room temperature 0 0 ~r X N (CH 2 )n N N H H 2H~r 1i BNIPDapen, n= 3 Ill BNIPDahex, n= 4 IV BNIPDahep, n = 5 V BNIPDaoct, n = 6 VI BNIPDanon, n = 7 VII BNIPDadec, ri = 8 Vill BNIPDadod, n = 10 [24] Scheme 2. Synthesis of Bisnaphthalimidopropylalkyltriamines.

WO 2008/007262 PCT/IB2007/052311 8 H I H Mts Mts MtsN N N't H H 1. DMF, Cs 2

CO

3 for 12 hr at 4 0 *0 2. CH 2 01 2 , 20% HBrigaicial CH00 OH BN PD pta Vernight at roorntemperature 00

IX

H H H O 3HBr 0 BNIPDeta 00 0 H x -- H H \HBr \ / [25] Biological Activities [26] The in vitro cytotoxicity of all the bisnaphthalmidopropyl derivatives described above were studied against colon cancer cell lines CaCo-2 and parasite Leishmania infantum. In the cancer cell line the IC 50 values of each compound were determined after 24 and 48 hr drug exposure (Table 1). All compounds except for BNIPDeta (IC 5 0 values, 21.7 and 22.3 !M for 24 and 48 h respectively exerted ICso values between 0.15 and 8.00 pM. BNIPSpd was the most active compound (IC 5 o, 0.15 and 0.47 iM at 24 and 48h respectively). In the same order of activity, the compound BNIPDadec showed a IC50 0.77 and 0.36 [M, after 24h and 48h of incubation, respectively. [27] The removal of a nitrogen atom from the linker chain does not appear to sub stantially affect the cytotoxic properties of these compounds. We previously reported that when the central alkyl group is a butyl chain, the compound (BNIPPut) is not soluble in most solvents and the aqueous solubility of bisnapthalimidopropyl compounds is enhanced by introducing a heteroatom like nitrogen in the central chain (Kong et aL, 2000). Here, by increasing the length of the alkyl central chain such as in BNIPDao, BNIPDan and BNIPDad, also helps aqueous solubility. We reason that with the longer alkyl chain, the two naphthalimido rings do not tend to stack on top of each other by T-T interactions between the aromatic rings and hence favour aqueous solubility. Among the latter compounds, BNIPDad showed the highest cytotoxicity against Caco-2 cells with ICo values of 0.36 pM (48 hr) and 0.77 pM (24 hr). [28] Table 1: Cytotoxicity of polyamine analogues against CaCo-2 cancer cells Compound- IC 50 (jiM) 24h 48h BNIPSpd 0.15 0.47 WO 2008/007262 PCT/IB2007/052311 9 BNIPPut ND ND BNIPDapen 11.00 6.50 BNIPDahex 0.65 2.00 BNIPDahep 3.20 0.94 BNIPDaoct 6.20 3.20 BNIPDanon 3.60 0.67 BNIPDadec 0.77 0.36 BNIPDadod 4.50 2.70 BNIPDpta 5.00 3.20 BNIPDeta 21.70 22.30 a Cytotoxicity determined by MTT assay. Data obtained after treating Caco-2 cells with varying concentrations of analogues (0.01-40 pM) for 24 and 48 hours. Data are mean ± SD of 6 replicates. ND: not determined. [29] Figure 1. The growth curve of luciferase promastigote and axenic amastigote forms. In vitro effect of different bisnaphthalimidopropil derivatives on parasite growth. The results are representative of 5 assays made independently. The pro mastigote and ax6nic amastigote were incubated with a serial range of drug concen trations 0.30 to 12.5 [LM, at 27 'C and 3.125 to 100 pM at 37 'C in 5% C0 2 , re spectively, during 3 days. The growth curves represented indicate the percentage growth related to the control for each concentration after luciferase activity de termination. Each point represents a mean of 3 assays t STD. [30] The treatment of different forms of the parasite Leismania infantum, promastigote, axenic amastigote and intracellular amastigote, with the compounds, BNIPSpd, BNIPPut, BNIPDaoct, BNIPDanon, BNIPDpta, BNIPDeta, in range of concentration 0.39 to 12.50 pM resulted in a dose dependent inhibition of parasite growth, except to the axenic amastigote incubated with BNIPDaoct, which didn't inhibited the parasite growth up to 50 pM concentration (Figure 1). We have observed that the parasite growth was completely blocked after 6.25 M to the promastigote and 50 pM to the axenic amastigote, except to the BNIPDaoct. [31] In the case of promastigote, the IC50± SD determined were 1.86±0.82, 0.40±0.15, #0.39, 2.09±0.54, 1.09±0.12, 0.96±0.17, for BNIPSpd, BNIPPut, BNIPDaoct, BNIPDanon, BNIPDpta, BNIPDeta, respectively. The most active compound for this parasite form was BNIPDoct. [32] In case of axenic amastigote form the IC50± SD determined were 9.61±1.84, 5.49±0.67, #50,00, 17.42±0.97, 5.24±0.93, 6.97±0.20 riM, for the following compounds BNIPSpd, BNIPPut, BNIPDaoct, BNIPDanon, BNIPDpta, BNIPDeta, re- WO 2008/007262 PCT/IB2007/052311 10 spectively. The most active compound for this parasitic form was BNIPDpta. [33] In the case of intracellular amastigote form the IC50± SD determined were 8.921.07, 4.53+0.54, 2.43±0.19, 6.03±0.67, 4.22±1.07, 9.52+0.56 pM, for the following compounds, BNIPSpd, BNIPPut, BNIPDaoct, BNIPDanon, BNIPDpta, BNIPDeta, respectively. The most active compound for this parasitic form was BNIPDaoct. [34] According to the results obtained all the compounds in study have anti-parasitic activity that give potential drugs to the leishmaniase treatment. [35] Table 2. Cytotoxicity of bisnaphthalimidopropil derivative compounds in different forms of the parasite Leishmania infantum

IC

5 0 (ttM) Compounds a Promastigotes Amastigotes Amastigote axenic intracellular BNIPSpd 1.86 ± 0.82 9.61 1.84 8.92± 1.07 BNIPPut 0.40 0.15 5.49 ± 0.67 4.53 ± 0.54 BNIPDapen ND ND ND BNIPDahex ND ND ND BNIPDahep ND ND ND BNIPDaoct # 0.39 # 50.00 2.43±0.19 BNIPDanon 2.09 ± 0.54 17.42 t 0.97 6.03 ± 0.67 BNIPDadec ND ND ND BNIPDadod ND ND ND BNIPDpta 1.09 ±0.12 5.24± 0.93 4.22± 1.07 BNIPDeta 0.96 0.17 6.97 0.20 9.52 0.56 Cytotoxicity determined by luciferase aasay. The results were obtained after treatment of different parasite forms with a range of different drug concentrations 0.30 to 100 pM after 72h of incubation. The results were representative of medium ± SD at least 5 assays. ND: not determined. [36] In conclusion, the new bisnaphthalimidoprpyl derivatives exhibit cytotoxicity that may be further developed as anti-tumour and/or anti-parasitic therapeutic agents. Conclusion [37] The use of the compounds in formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI could be an advantage in the treatment of cancer and parasitic disease namely, treatment of leishmaniasis, trypanosomiasis and malaria.

WO 2008/007262 PCT/IB2007/052311 11 [38] For preparation of the pharmaceutical compositions with the compounds of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, a inert pharmaceutical adjuvant are mixed with active compounds. The adjuvant used could be solid or liquid. The solid forms include powder, pill, grainy disperse and capsule. The solid adjuvant could be one or more substance that could be diluents, flavouring agents, sweeteners, solvents, lubricants, suspension agents, binding agents or disaggregating agents and could be a encapsulating agents. [39] The pharmaceutical preparation is preferentially presented on single dose form, the package contains discrete quantities of the preparation as cover pills, capsules, power in flask or ampoule and liposome formula. [40] The dose could range according to the needs of the animal or the patient, the severity of the disease and the compound to be used. The determinations of the dose for a particular situation regards to the people skilled in the art. For convenience, the total daily dose could be devised and distributed administration during the day. Detailed description of the invention [41] General method for the synthesis of mesitylated di or triamine [42] Corresponding diamine or triamine was dissolved in anhydrous pyridine followed by the addition of mesitylene chloride (2.1 molar excess for diamine and 3.1 molar excess for triamine). The resulting solution was stirred at room temperature for 4 hours. Removal of the pyridine followed by the addition of cold water resulted in the formation of a precipitate. The latter was filtered off and washed thoroughly with water. The crude product was recrystallised from absolute ethanol. [43] NI,NI-Dimesityloctane 2- (70%), 13 C NMR (CDCl 3 ) d: 20.82 (CH 3 , Mts), 22.85

(CH

3 , Mts), 26.34 (CH 2 ), 28.70 (CH 2 ), 29.41 (CH2), 41.05 (N-CH 2 ), 47.58 (CH 2 ), 133 (Aromatic carbons, Mts). [44] NI,N 9 -Dimesitylnonane 3- (36%), 13 C NMR (CDCl 3 ) 11: 20.82 (CH 3 , Mts), 22.85

(CH

3 , Mts), 26.34 (CH 2 ), 28.70 (CH 2 ), 29.41 (CH 2 ), 41.05 (N-CH 2 ), 47.58 (CH 2 ), 133 (Aromatic carbons, Mts). [45] NI,N1O-Dimesityldecane 4- (48%), 13 C NMR (CDCl 3 ) d: 20.82 (CH 3 , Mts), 22.85

(CH

3 , Mts), 26.34 (CH 2 ), 28.70 (CH 2 ), 29.41 (CH 2 ), 41.05 (N-CH 2 ), 47.58 (CH 2 ), 133 (Aromatic carbons, Mts). [46] NI,N, N9-Trimesityldipropyltriamine 5- (67%), I 3 C NMR (CDCl 3 ) 4: 20.85 (CH 3 , Mts), 22.79 (CH 3 , Mts), 27.69 (CU 2 ), 39.50 (N-CH 2 ), 43.11 (N-CH 2 ), 132.17 (Aromatic carbons, Mts), 139.98 (Aromatic carbons, Mts). [47] NI,N 3 , N6-Trimesityldiethyltriamine 6- (59%), 13C NMR (CDCl 3 ) d: 21.35 (CH 3 , Mts), 23.06 (CH 3 , Mts), 41.05 (N-CH 2 ), 47.58 (N-CH 2 ), 133 (Aromatic carbons, Mts). [48] Synthesis of O-tosylpropylnaphthalimide [49] Naphthalic anhydride (6.34g, 0.032 mol) was dissolved in DMF (50 mL) followed by the addition of aminopropanol 3 (2.45g, 0.032 mol) and DBU (4.87g, 0.032 mol). The solution was left stirring at 85'C for 4 hr. The solvent DMF was removed under WO 2008/007262 PCT/IB2007/052311 12 reduced pressure and the resulting residue was poured into cold water with stirring (200 mL) to form a precipitate. The latter was filtered using a buchner funnel and washed thoroughly with (i) water (ii) saturated bicarbonate solution. The yield of the reaction was found to be 95%. This compound, Naphthalimidopropanol was pure enough and was used in the next step with no further purification. NMR (CDC1 3 ): 4 = 8.65-7.80 (in, 6H, aromatic protons), 4.39 (t, 2H, -N-CH 2 ), 3.69 (t, 2H, CH 2 -O-), 3.20 (s. broad, 1 H , OH), 2.06 (p, 2H, CH 2 ). 13C NMR (CDCl 3 ): d = 161.70 (C=0), 135.70-122.90 (aromatic carbons), 74.90, 59.90, 30.90 (3 xCH 2 ). [50] Naphthalimidopropanol (5. 1Og, 20 mmol) was dissolved in anhydrous pyridine (80 mL). The solution was stirred for 15 mins at 4 'C. Tosyl Chloride (5.72g, 30 mmol) were added, in small portions, over 30 mins . The solution was left overnight at 4'C and was poured into ice water (200 mL) to form a solid on standing. The solid formed was filtered off and washed thoroughly with water. The crude product was recrys tallised from either ethanol or ethylacetate to give O-tosylpropylnaphthalimide 6 (53%). 1 H NMR (CDCl 3 ): 4 = 8.65-7.80 (in, 6H, aromatic protons), 4.45 (t, 2H, CH 2 ), 4.35 (t, 2H, CH 2 ), 2.50 (s, 3H, CH 3 ), 2.25 (p, 2H, CH 2 ). 1 3 C NMR (CDCl 3 ): d = 161.30 (C=O), 145.10-123.10 (aromatic carbons), 73.10, 67.90, 28.70 (3 x CH 2 ), 22.10 (CH 3 ). LRMS (FAB): Caled. for C 2

H

19 N0 6 S 425.09, Found: 426 [MH]+. [51] General N-alkylation Reaction (Step I in scheme 1) [52] Mesitylated polyamines (2-6) (0.651 mmol) were dissolved in anhydrous DMF (13.5 mL) followed by the addition of 7 (0.13 mmol) and cesium carbonate (1.06g). The solution was left at 85'C and completion of the reaction was monitored by thin layer chromatography. DMF was removed under vacuo and the residue was poured into cold water and the resulted precipitate filtered and washed thoroughly with water. After drying the crude was recrystallised from ethanol to give the fully protected pure product in high yield (75-85%). [53] General Deprotection Reaction (step 2 in scheme 1) [54] The fully protected polyamine derivatives (0.222 mmol) were dissolved in anhydrous dichloromethane (10 mL) followed by the addition of hydrobromic acid/ glacial acetic acid (1 mL). The solution was left stirring at room temperature for 24 h. The yellow precipitate formed, was filtered off and washed with dichloromethane, ethylacetate and ether. [55] Using the process described and related processes, currently used by the ones skilled in the art, using alkyl chain appropriated, were synthesized, - Bisnaphthalimidopropyldiaminopentane (BNIPDapen. LRMS (ESI): calc C 3 5 H3 6

N

4 0 4 2HBr 738.52 [M]+, found: 657.1 [M-H-2Br]+. - Bisnaphthalimidopropyldiaminohexane (BNIPDahex). LRMS (ES): calc C 36

H

38

N

4 0 4 2HBr 752.71 [M]+, found: 671.3[M-H-2Br]+. - Bisnaphthalimidopropyldiaminoheptane (BNIPDahep). RMS (ESI): calc C 3 7 H 4oN 4 0 4 2HBr 766.74 [M]+, found: 671.3[M-H-2Br]+.

WO 2008/007262 PCT/IB2007/052311 13 Bisnaphthalimidopropyldiaminooctane - (BNIPDaoct) (85%), DMSO-d 6 , 4: 24.43 (CH 2 ), 25.30 (CH 2 ), 25.66 CH 2 ), 28.07 (CH 2 ), 44.72 (N-CH 2 ), 46.60

(N-CH

2 ), 121.99, 127.13, 130.62, 131.21, 134.31 (Aromatic Carbons), 163.61 (C=O). LRMS (FAB): Caled. for C 3 sH 42

N

4 0 4 H 619.3279, Found: 619.3282 [M\-H-2Br]+. Bisnaphthalimidopropyldiaminononane(BNIPDanon) (85%), DMSO-d 6 , .: 24.88 (CH 2 ), 25.84 (CH 2 ), 26.16 CH 2 ), 28.76 (CH 2 ), 45.29 (N-CH 2 ), 47.29

(N-CH

2 ), 121.94, 127.51, 131.12, 131.42, 134.76 (Naphthalimido aromatic Carbons), 164.00 (C=O). LRMS (FAB): Caled. for C3 9

U

44

N

4 0 4 H 633.3435, Found: 633.3440 [M-H-2Br]+. Bisnaphthalimidopropyldiaminodecane(BNIPDadec) (75%), DMSO-d 6 , 41: 24.97 (CH 2 ), 25.90 (CH 2 ), 26.22 CH 2 ), 28.79 (CH 2 ), 29.00 (CH 2 ), 45.35

(N-CH

2 ), 47.38 (N-CH 2 ), 121.05, 127.66, 131.30, 131.51, 134.94 (Naphthaliniido aromatic Carbons), 164.21 (C=O). LRMS (FAB): Calcd. for

C

40 H4 6

N

4 0 4 H 647.4, Found: 647.4 [M-H-2Br]+. - Bisnaphthalimidopropyldiaminododecane (BNIPDadod). LRMS (FAB): Caled. for C 4 2 HoN 4 0 4 H 836.71, Found: 675.4 [M-2H-2Br] . - Bisnaphthalimidopropyldipropiltriamine(BNIPDpta) (85%), DMSO-d 6 , 4: 22.20 (CH 2 ), 24.70 (CH 2 ), 44.10 (N-CU 2 ), 44.20 (N-CH 2 ), 45.00 (N-CH 2 ), 130 (Aromatic Carbons) 164.87 (C=O). LRMS (FAB): Caled. for C 3 6

H

39

N

5 0 4 3HBr 850.7, Found: 606.4 [M-2H-3Br]+. - Bisnaphthalimidopropyldietiltriamine (BNIPDeta) (67%), DMSO-d 6 , d: 22.20 (CH 2 ), 24.70 (CU 2 ), 44.10 (N-CH 2 ), 44.20 (N-CH 2 ), 45.00 (N-CH 2 ), 130 (Aromatic Carbons). LRMS (FAB): Calcd. for C 34

H

35

N

5 0 4 3HBr 820.40, 578.2762 [M-2H-3Br]+. Found: 578.2760 [M-2H-3Br]*. Example 1: Synthesis of BNIPDaoct [56] The diamine was dissolved in anhydrous pyridine followed by the addition of mesitylene chloride (2.1 molar excess). The resulting solution was stirred at room tem perature for 4 hours. Removal of the pyridine followed by the addition of cold water resulted in the formation of a precipitate. The latter was filtered off and washed thoroughly with water. The crude product was recrystallised from absolute ethanol. [57] NI,N-Dimesityloctane 2- (70%), 13 C NMR (CDCl 3 ) 4: 20.82 (CH 3 , Mts), 22.85

(CH

3 , Mts), 26.34 (CH 2 ), 28.70 (CH 2 ), 29.41 (CH 2 ), 41.05 (N-CH 2 ), 47.58 (CH 2 ), 133 (Aromatic carbons, Mts). [58] The naphthalic anhydride (6.3 4 g, 0.032 mol) was dissolved in DMF (50 mL) followed by the addition of aminopropanol 3 (2.45g, 0.032 mol) and DBU (4.87g, 0.032 mol). The solution was left stirring at 85oC for 4 hr. The solvent DMF was removed under reduced pressure and the resulting residue was poured into cold water with stirring (200 mL) to form a precipitate. The latter was filtered using a buchner funnel and washed thoroughly with (i) water (ii) saturated bicarbonate solution. The WO 2008/007262 PCT/IB2007/052311 14 yield of the reaction was found to be 95%. This compound, Naphthalimidopropanol was pure enough and was used in the next step with no further purification. NMR

(CDC

3 ): d = 8.65-7.80 (m, 6H, aromatic protons), 4.39 (t, 2H, -N-CH 2 ), 3.69 (t, 2H,

CH

2 -O-), 3.20 (s, broad, 1 H , OH ), 2.06 (p, 2H, CH 2 ). 13C NMR (CDC1 3 ): d = 161.70 (C=O), 135.70-122.90 (aromatic carbons), 74.90, 59.90, 30.90 (3 xCH 2 ). [59] Naphthalimidopropanol (5. 1Og, 20 mmol) was dissolved in anhydrous pyridine (80 mL). The solution was stirred for 15 mins at 0 'C. Tosyl Chloride (5.72g, 30 mmol) were added, in small portions, over 30 mins. The solution was left overnight at 4'C and was poured into ice water (200 mL) to form a solid on standing. The solid formed was filtered off and washed thoroughly with water. The crude product was recrys tallised from either ethanol or ethylacetate to give O-tosylpropylnaphthalimide 6 (53%). 1 H NMR (CDCl 3 ): d = 8.65-7.80 (in, 6H, aromatic protons), 4.45 (t, 211, CH 2 ), 4.35 (t, 2H, CH 2 ), 2.50 (s, 3H, CH 3 ), 2.25 (p, 2H, CU 2 ). 13C NMR (CDCL 3 ): d = 161.30 (C=O), 145.10-123.10 (aromatic carbons), 73.10, 67.90, 28.70 (3 x CH 2 ), 22.10 (CH 3 ). LRMS (FAB): Caled. for C 1 2 HqNO 6 S 425.09, Found: 426 [MH]+. [60] The mesitylated polyamines (0.651 mmol) were dissolved in anhydrous DMF (13.5 mL) followed by the addition of 7x (0.13 miol) and cesium carbonate (I.06g). The solution was left at 85'C and completion of the reaction was monitored by thin layer chromatography. DMF was removed under vacuo and the residue was poured into cold water and the resulted precipitate filtered and washed thoroughly with water .After drying the crude was recrystallised from ethanol to give the fully protected pure product in high yield (75-85%). [61] The fully protected polyamine derivatives (0.222 mmol) were dissolved in anhydrous dichloromethane (10 mL) followed by the addition of hydrobromic acid/ glacial acetic acid (1 mL). The solution was left stirring at room temperature for 24 h. The yellow precicipate formed, was filtered off and washed with dichloromethane, ethylacetate and ether. [62] By this way were synthesized the The Bisnaphthalimidopropyldiaminooctane (BNlPDaoct) (85%), DMSO-d 6 , d :24.43 (CH 2 ), 25.30 (CH 2 ), 25.66 CH 2 ), 28.07 (CH2 ), 44.72 (N-CH 2 ), 46.60 (N-CH 2 ), 121.99, 127.13, 130.62, 131.21, 134.31 (Aromatic Carbons), 163.61 (C=O). LRMS (FAB): Calcd. for C 3 sH 42

N

4 0 4 H 619.3279, Found: 619.3282 [M-H-2Br]+. Example 2: Synthesis of BNIPDpta [63] The dipropiltriamine was dissolved in anhydrous pyridine followed by the addition of mesitylene chloride (3.1 molar excess). The resulting solution was stirred at room temperature for 4 hours. Removal of the pyridine followed by the addition of cold water resulted in the formation of a precipitate. The latter was filtered off and washed thoroughly with water. The crude product was recrystallised from absolute ethanol. [64] N,N, N9-Trimesityldipropyltriamine 5- (67%), 3C NMR (CDCl 3 ) d : 20.85 (CH 3 , Mts), 22.79 (CH 3 , Mts), 27.69 (CH 2 ), 39.50 (N-CH 2 ), 43.11 (N-CH 2 ), 132.17 WO 2008/007262 PCT/IB2007/052311 15 (Aromatic carbons, Mts), 139.98 (Aromatic carbons, Mts). [65] The naphthalic anhydride (6.34g, 0.032 mol) was dissolved in DMF (50 mL) followed by the addition of aminopropanol 3 (2.45g, 0.032 mol) and DBU (4.87g, 0.032 mol). The solution was left stirring at 85-C for 4 hr. The solvent DMF was removed under reduced pressure and the resulting residue was poured into cold water with stirring (200 mL) to form a precipitate. The latter was filtered using a buchner funnel and washed thoroughly with (i) water (ii) saturated bicarbonate solution. The yield of the reaction was found to be 95%. This compound, Naphthalimidopropanol was pure enough and was used in the next step with no further purification. NMR (CDCl 3 ): d = 8.65-7.80 (in, 6H, aromatic protons), 4.39 (t, 211, -N-CH 2 ), 3.69 (t, 2H,

CH

2 -O-), 3.20 (s, broad, 1 H , OH ), 2.06 (p, 2H, CH 2 ). 3C NMR (CDCl 3 ): d = 161.70 (C=O), 135.70-122.90 (aromatic carbons), 74.90, 59.90, 30.90 (3 xCH 2 ). [661 Naphthalimidopropanol (5.1Og, 20 mmol) was dissolved in anhydrous pyridine (80 mL). The solution was stirred for 15 mins at 4 'C. Tosyl Chloride (5.72g, 30 minol) were added, in small portions, over 30 mins . The solution was left overnight at 4 "C and was poured into ice water (200 mL) to form a solid on standing. The solid formed was filtered off and washed thoroughly with water. The crude product was recrys tallised from either ethanol or ethylacetate to give 0-tosylpropylnaphthalimide 6 (53%). 1 H NMR (CDCl 3 ): d = 8.65-7.80 (m, 611, aromatic protons), 4.45 (t, 2H, CH 2 ), 4.35 (t, 2H, CH 2 ), 2.50 (s, 3H, CH 3 ), 2.25 (p, 2H, CH 2 ). 13C NMR (CDCl 3 ): d = 161.30 (C=O), 145.10-123.10 (aromatic carbons), 73.10, 67.90, 28.70 (3 x CH 2 ), 22.10 (CH 3 ). LRMS (FAB): Caled. for C 12

H

19 N0 6 S 425.09, Found: 426 [MH]+. [67] The mesitylated polyamines (0.651 mmol) were dissolved in anhydrous DMF (13.5 mL) followed by the addition of 7x (0.13 mmol) and cesium carbonate (1.06g). The solution was left at 85 "C and completion of the reaction was monitored by thin layer chromatography. DMF was removed under vacuo and the residue was poured into cold water and the resulted precipitate filtered and washed thoroughly with water. After drying the crude was recrystallised from ethanol to give the fully protected pure product in high yield (75-85%). [68] The fully protected polyamine derivatives (0.222 mmol) were dissolved in anhydrous dichloromethane (10 mL) followed by the addition of hydrobromic acid/ glacial acetic acid (1 mL). The solution was left stirring at room temperature for 24 h. The yellow precipitate formed, was filtered off and washed with dichloromethane, ethylacetate and ether. [69] By this way were synthesized the Bisnaphthalimidopropyl dipropiltriamine(BNIPDpta) (85%), DMSO-d 6 , d: 22.20 (CH 2 ), 24.70 (CH 2 ), 44.10 (N-CH2), 44.20 (N-CH 2 ), 45.00 (N-CH 2 ), 130 (Aromatic Carbons) 164.87 (C=O). LRMS (FAB): Caled. for C 36

H

39

N

5 0 4 3HBr 850.7, Found: 606.4 [M-2H-3Br]+. Bibliography [70] WO 2008/007262 PCT/IB2007/052311 16 1. Brana, M.F.; Ramos, A.. Curr. Med. Chem. Anti-Cancer Agents. 2001, 1, 237. 2. Kong Thoo Lin, P.; Pavlov, V. A. Bioorganic and Medicinal Chemistry Letters, 2000, 10, 1609. 3. Pavlov, V. A.; Kong Thoo Lin, P.; Rodilla, V. Chemico-Biological In teractions, 2001, 137, 15. 4. Patten, A.D.; Sun, J-H; Ardecky, R.J. U.S. Patent, 5,086,059, 1992. 5. Brana, M.F.; Castellano, J.M.; Moran, M.; Perez de Vega, M.J.; Qian, X.D.; Romerdahl, C.A.; Kcilhauer, G. European Journal ofMedicinal Chemistry, 1995, 30, 235. 6. Bailly, C.; Carrasco, C.; Joubert, A.; Bal, C.; Wattez, N.; Hildebrand, M-P.; Lansiaux, A.; Colson, P.; Houssier, C.; Cacho, M.; Ramos, A.; Brana, M.F. Biochemistry, 2003, 42, 4136. 7. Carrasco, C.; Joubert, A.; Tardy, C.; Maestre, N.; Cacho, M.; Brana, M.F.; Bailly, C. Biochemistry, 2003, 42, 11751. 8. Kong Thoo Lin, P.; Dance, A.M.; Bestwick, C.; Milne, L. Biochemical Society Transactions, 2003, 31, 407. 9. Pavlov, V.A.; Rodilla, V.; Kong Thoo Lin, P. Life Sciences, 2002, 71, 1161. 10. Dance, A.M.; Ralton, L.; Fuller, Z.; Milne, L.; Duthie, S.; Bestwick, C.S.; Kong Thoo Lin, P. Biochemical Pharmacology, 2005, 69,19 11. Tavares, J.; Quaissi, A.; Kong Thoo Lin, P.; Tomas, A.; Cordeiro-da-Silva, A. International Journalfor Parasitology,2005, 35, 637. 12. Murry, H.W. Antimicrobial Agents and Chemotherapy, 2001, 45, 2185. 13. Muller, S.; Coombs, G.H.; Walter, R.D. Trends Parasitology, 2001, 17, 242. 14. Fairlamb, A.H.; Cerami, A.Annual Review Microbiology, 1992, 46, 695. 15. Barrett, M.P.; Mottram, J.C.; Coombs, G.H. Trends in Microbiology1999, 7, 82. 16. Thoo Lin, P.K. and. Pavlov, V.A. Bioorganic and Medicinal Chemistry Letters,1998, 10, 1609. 17. Roy, G.; Dumas, C.; Sereno,D.; Wu,Y.; Singh, A.K.; Tremblay, M.J.; Ouellette, M.; Olivier, M.; and Papadopoulou, B. Molecular and Biochemical Parasitology,2000, 110, 195.

Claims (1)

1. Claims

   [1 ] The compound from the formula A,

   characterized in that the atkyl chain that binds the two aromatic rings changes in length, between 4 and 20 carbon atoms and the number of nitrogen atoms, between 2 and 6. [2] Compounds as defined in claim 1, characterized in that:

   Bisnaphthalimidopropyldiaminopentane - BNIPDapen (II) Bisnaphthalimidopropyldiaminohexane - BMPDahex (III) Bisnaphthalimidopropyldiaminoheptane - BNIPDahep (IV) Bisnaphthalimidopropyldiaminooctane - BNIPDaoct (V) Bisnaphthalimidopropyldiaminononanc - BNEPDanon (VI) Bisnaphthalimidopropyldiaminodecane - BNIPDadec (VII) Bisnaphthalimidopropyldiaminododecane-BNIPDadod (VIII) Bisnaphthalimidopropyldipropiltriamine - BNIPDpta (IX) Bisnaphthalimidopropyldietiltriamine-BNIPDeta (X). [3] Pharmaceutical composition used in treatment of cancer and parasitic disease, namely cancer treatment, trypanosomiasis treatment, leishmaniasis treatment and malaria treatment, characterized in that it includes as active ingredient, at least one compound according claim 1 and/or 2.

   [4] Pharmaceutical composition, characterized in that it contains one compound of the formula A and derivatives II, m, IV, V, VI, VTI, VIII, IX, X thereof, in association with a vehicle or excipient pharmaceutically accepted.

   [5] Pharmaceutical composition, according to claims 4 and 5, characterized in that it includes at least one of different compounds used on prevention and /or disease treatment involving the infections by protozoa, such as, leishmaniasis, trypanosomiasis and malaria, such compounds including miltefosinc, penthavalent an- tymonial derivates, amphotericyne B, pentamidine and derivates, melarsoprol, benzimnidazol, nifurtimox, ketoconazol, difluorometilornitine, cloroquine and their derivates and quinine. [6] Pharmaceutical composition, according to 4 and 5, characterized in that it includes at least one of different compounds used on the preparation and / or treatment of cancer disease.

   [7] Use of pharmaceutical preparation according to claims 4 and 6, characterized in that it is directed to parasitic disease treatment induced by the genus parasite Leishmania, including the species, L. infantum, L. donovani, L. major. L. tropica,

   L. mexicana, L. amazonensis and L. braziliensis . [8] Use of the pharmaceutical preparation according to claims 4 and 6, characterized in that it is directed to parasitic disease treatment induced by the genus parasite

   Trypanosoma, including the species, T. cruzi, T. brucei, T. gambiense. [9] Use of the pharmaceutical preparation according to claims 4 and 6, characterized in that it is directed to parasitic disease treatment induced by the genus parasite

   Pasmodium, included the species, P. falciparum, P. vivax, P. ovale. [10] Use of the pharmaceutical preparation according to the claim 7, characterized in that it is directed to cancer disease treatment and / or prevention.